Type 1 diabetes (T1D; also known as type 1 diabetes mellitus and immune-mediated diabetes) is a multifactorial autoimmune disease in which insulin-producing beta cells in pancreatic islets are destroyed by autoreactive T cells. Mononuclear cells infiltrate the pancreatic islets of Langerhans during a variable period of clinically silent inflammation (insulitis), and eventually T cells destroy insulin-producing beta cells. Full-blown type 1 diabetes ensues when most beta cells are destroyed and the pancreas ceases to produce enough insulin. Exogenous insulin must then be administered for life. Weeks or months after insulin treatment starts, patients with type I diabetes can experience a variable period of remission, which is thought to result from restored insulin production by residual beta cells. Continued treatment with exogenous insulin is needed to preserve the residual beta cells, which can still naturally modulate glucose metabolism.
Type 2 diabetes is characterized by insulin resistance which may be combined with reduced insulin secretion. The defective responsiveness to insulin is believed to involve the insulin receptor. In the early stage of type 2 diabetes, hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver. As the disease progresses, impairment of insulin secretion occurs, and therapeutic replacement of insulin is often required. Beta cell destruction also occurs in type 2 diabetes, and it has been proposed that one contributing factor is increased beta cell apoptosis (Rhodes Science 2005 Jan. 21; 307(5708):380-4).
A widely used model of autoimmune T1D is the non-obese diabetic (NOD) mouse, which develops diabetes spontaneously after a variable period of insulitis, similarly to human T1D. NOD mice demonstrate insulitis from 4-5 weeks of age, and after a variable period of chronic inflammation, diabetes develops about 10-20 weeks later, with most females diabetic by 30 weeks of age (1, 2). An additional accepted model of experimental autoimmune diabetes in mice is the induction of diabetes by multiple injections of low doses of streptozotocin (LDST) (3, 4). Streptozotocin causes diabetes by direct beta cell cytotoxicity, as well as by initiation of cell mediated autoimmune reaction against beta cells (4, 5). Adoptive transfer of activated splenocytes from LDST-treated mice has been disclosed to induce diabetes in untreated healthy mice (5).
T1D is considered to be a T cell mediated disease. However, several studies suggest that the innate immune system, in particular natural killer (NK) cells, play a role in the pathogenesis of the disease. For example, it has been disclosed that NK cells infiltrate the islets of NOD mice (7), and islet inflammation mediated mainly by NK cells has been reported in human T1D (8). Several alterations in NK cell compartments in patients with T1D have been disclosed, both at the onset of the disease and after long term hyperglycemia (9). Furthermore, the essential role of NK cells in diabetes development was demonstrated in murine models of accelerated T1D (10, 11). Poirot et al., 2004 disclose that the proportion and number of NK cells, and the timing of their entry to the pancreas correlate with the severity of T1D in transgenic NOD mice (10). It has also been disclosed that depletion of NK cells in transgenic NOD mice models of accelerated T1D significantly inhibits diabetes development (10, 11). However, the molecular mechanisms of NK cell involvement in T1D are still unknown.
NK cells play a crucial role in the initial defense against virus-infected cells and cancer cells (12). They interact with antigen presenting cells (APCs), serve as APCs, directly kill hazardous cells and further secrete chemokines and immunomodulatory cytokines such as IFN-γ and TNF-α, which cause T cells to shift into a Th1 phenotype (13, 14).
NK cells recognize target cells through a diverse array of activating receptors and a delicate balance between inhibitory and activating signals tightly regulates their activation (15-17). NK cells have been identified in target organs of patients suffering from autoimmune diseases (18) and they are capable of attacking autologous cells (19-21).
The killing mediated by NK cells involves several activating receptors, such as the natural cytotoxicity receptors (NCRs) NKp30, NKp44 and NKp46, and NKG2D. NKp30, NKp44 and NKp46 are expressed almost exclusively on NK cells, whereas NKG2D is expressed in additional types of lymphocytes such as CD8+ T cells (22). NKp46 is considered to be the most specific NK marker for which an ortholog protein (NCR-1) has been found in mice (23, 24).
Pessino et al., 1998 discloses molecular cloning of NKp46 and its role in mediated cytotoxicity (Pessino et al., J Exp Med 1998; 188:953-960).
Some of the inventors of the present invention have disclosed that soluble NKp46- and NKp44-immunoglobulin (Ig) fusion proteins, but not an NKp30-Ig fusion protein, specifically bind to hemagglutinin of influenza virus and to hemagglutinin-neuraminidase of Sendai virus (29, 45, 46). According to these disclosures, this interaction is functional and can mediate an enhanced killing of infected cells. Furthermore, the enhanced killing can be abolished by antibodies that block either the HA or the receptors NKp46 and NKp44.
The human NKp46 receptor has multiple isoforms including isoform a (GenBank Accession No. CAA04714); isoform b (GenBank Accession No. CAA06872); isoform c (GenBank Accession No. CAA06873), and isoform d (GenBank Accession No. CAA06874). In general the NKp46 receptor comprises two extracellular Ig-like domains of the C2 type, a transmembrane portion and an intracellular segment. The extracellular portion of NKp46 comprises a D1 domain, designated NKp46D1 (corresponding to residues 22-120 of the mature full length protein of isoform a), and a D2 domain, designated NKp46D2, comprising 134 amino acid residues (corresponding to residues 121-254 of the full length protein of isoform a).
PCT Application Publication No. WO 02/08287 of some of the present inventors discloses a targeting complex comprising a target recognition segment comprising one of NKp30, NKp44 and NKp46 or a functional fragment thereof; and an active segment comprising an active substance such as a cytotoxic moiety, an imaging moiety or an Ig fragment. According to the disclosure, fusion proteins containing the extracellular domains NKp30, NKp44 or NKp46 fused to the Fc portion of human IgG1 (termed respectively NKp30-Ig, NKp44-Ig and NKp46-Ig), bind certain tumor cell targets, and NKp46-Ig binds to virus infected cells. Further disclosed are fusion proteins containing either D1 or D2 fused to the Fc portion of human IgG1 (termed respectively NKp46D1-Ig and NKp46D2-Ig), and the observation that D2 is responsible for interaction with viral hemagglutinin.
PCT Application Publication No. WO 2004/053054 of some of the present inventors discloses that an NKp30-Ig conjugate is effective in inducing tumor regression in vivo in cancer bearing nude mice. Further disclosed are pharmaceutical compositions comprising a first segment selected from NKp30, NKp44 and NKp46 or a functional fragment thereof, and a second segment selected from an Ig molecule or a fragment or Fc fragment thereof, for eliminating a tumor or inhibiting growth of a tumor.
PCT Application Publication No. WO 2005/000086 of some of the present inventors discloses isolated peptide fragments comprising glycosylated residues derived from NKp44 and NKp46 that comprise epitopes essential for binding to target cells. According to the disclosure, a linker peptide within the D2 domain of NKp46 designated NKp46LP, which corresponds to residues 215-254 of the full length protein, contains an O-glycosylated threonine residue that is essential for the binding of NKp46 to viral infected cells and to tumor cells. Further disclosed is a linker peptide derived from the extracellular domain of NKp44 which corresponds to residues 136-190 of the full length protein and comprises a hyper-glycosylated region comprising at least 14 predicted glycosylation sites that contribute to the efficient binding to viral-infected cells. Further disclosed are isolated peptide fragments of 10-100 amino acids, derived from the aforementioned peptides which retain the biological activity of interest.
PCT Application Publication No. WO 2005/051973 of some of the present inventors discloses peptides derived from NKp46, NKp44 and NKp30 which comprise sulfated polysaccharides and are capable of binding to tumor cells. Specifically disclosed are peptides derived from NKp46 corresponding to residues 153-172 and 153-175 of the full length protein; peptides derived from NKp30 corresponding to residues 57-84 and 57-76 of the full length protein, and a peptide derived from NKp44 corresponding to residues 51-74 of the full length protein
U.S. Patent Application Publication No. 2008/0274047 discloses methods of treating immunoproliferative and autoimmune disorders using antibodies which bind NK cell receptors, particularly to deplete cells involved in the immunoproliferative pathology. According to the disclosure, immmunoproliferative disorders which may be treated by the invention include type I diabetes, and the antibody may directed against human NKp46. Further disclosed is that injection of anti-human NKp46 antibodies into transgenic mice expressing human NKp46 resulted in depletion of NK cells in blood, spleen, liver and lung.
U.S. Patent Application Publication No. 2007/0231813 discloses methods and compositions to assess the therapeutic response of a subject to a therapeutic composition comprising an Fc portion, preferably a therapeutic antibody, wherein the therapeutic antibody preferably is not capable of, or is not required to be capable of, depleting target cells. According to the disclosure, the composition may specifically bind an NK receptor inter alia NKp46 and the subject may have juvenile onset diabetes.
U.S. Patent Application Publication No. 2004/0038339 discloses a multifunctional polypeptide comprising (a) a first domain comprising a binding site specifically recognizing an extracellular epitope of the NKG2D receptor complex; and (b) a second domain having receptor or ligand function, wherein said receptor or ligand function may be an antigen binding site of an antibody or fragment thereof directed against inter alia NKp46 which interacts with haemagglutinin (HA) of influenza virus. According to the disclosure, the composition may be used for treating autoimmune diseases, inter alia insulin-dependent diabetes mellitus, wherein elimination of the subpopulation of immune cells that causes the disease is desired.
PCT Application Publication No. WO 02/072631 discloses an MHC molecule construct comprising a carrier molecule having attached thereto one or more MHC molecules, and optionally further comprising one or more biologically active molecules inter alia NKp46. According to the disclosure, the construct may be used for prognosing or diagnosing a disease, or determining the effectiveness of a medicament against a disease, and the disease may be type I diabetes.
PCT Application Publication No. WO 2009/148568 discloses a cellular composition comprising at least about 30% human facilitating cells (hFCs) having a phenotype of CD8+/alpha beta TCR−/delta gamma TCR−/CD56dim/neg and wherein the hFCs optionally further have a phenotype including NKp46+. According to the disclosure, the composition may be used for transplantation into a human subject having a disease inter alia diabetes.
There remains an unmet need for therapeutic methods directed to preventing and inhibiting insulitis and subsequent onset of type I diabetes. The prior art does not teach or suggest using any of NKp46, an isolated fragment of NKp46 or a fusion protein comprising a fragment of NKp46 for suppressing development of type I diabetes.